SUMMARY/ABSTRACT Mammalian cells must respond to signals in their environment in complex ways in order to properly develop, form tissues, and marshal a defense against invading organisms. Much of the cell-cell communication needed is provided by a class of cell adhesion and signaling proteins that share a topology that includes an extracellular variable immunoglobulin-like domain, one or more constant immunoglobulin-like domains, a membrane anchor, and often a cytoplasmic domain that carries signaling elements, for example, CEACAMs (carcinoembryonic antigen related cell adhesion molecules) and Siglecs (Sialic acid-binding immunoglobulin- type lectins). The extra cellular domains of these molecules are heavily glycosylated and the glycans (carbohydrates) on these domains mediate or modify interactions with similar molecules on other cells and thus modulate biologically important signals. Understanding these interactions can provide opportunities to intervene when normal signaling and regulatory events fail as in the case of malignancy or when pathogens hijack interaction pathways to invade cells or evade immune response. Attaining this understanding has, however, been challenging because of the difficulty in dealing with the heterogeneity of glycans and the lack of methodology for the preparation and structural characterization of appropriately glycosylated samples. This project develops protein expression and glycan engineering technology to prepare molecular systems appropriate for structural characterization, and it develops NMR technology to carry out structural characterization. Development is driven primarily by application to terminal domains of CEACAMs and one of their well characterized glycan interaction partners, the carbohydrate recognition domain of DC-SIGN (Dendritic Cell-Specific Intracellular adhesion molecule 3 (ICAM-3)-Grabbing Nonintegrin). Their interaction is a well-recognized element in immune response and CEACAMs are among important biomarkers for cancer where variations in levels and glycosylation are believed to contribute to metastasis. To characterize interaction the following aims are pursued: (1) Properly glycosylated CEACAM domains are expressed in mammalian cells (HEK293) using recently developed expression and purification technology. (2) The expressed constructs are structurally characterized using long range constraints from NMR observation of a sparse set of isotopically labeled sites. (3) Glycans are reengineered to carry isotopically labeled epitopes known to interact with DC-SIGN. (4) NMR methodology is developed to assign resonances from multiple glycosylation sites and provide information to guide molecular dynamics simulations of glycan conformation and motion. And (5) the interaction energetics and conformation of DC-SIGN-CEACAM complexes are determined by NMR and SPR. It is hypothesized that relative domain orientations is key in the modulation of cell-cell interactions and the initiation of intracellular signals. Understanding how glycan interactions contribute to preferred orientation will open new opportunities for intervention in human disease.